Beclin 1 regulates neuroprotective TGF-beta signaling

描述（由申请人提供）：阿尔茨海默病（AD）仅在美国就影响超过500万人，但我们对发病机制的理解仍然不完整，目前没有治愈方法。最近，我们报道了自噬相关蛋白beclin 1在AD脑提取物中的水平降低。在小鼠中beclin 1的杂合缺陷导致突触丢失和神经变性。小胶质细胞中beclin 1水平的降低损害了吞噬细胞受体CD36的再循环，导致淀粉样蛋白斑块的清除减少，淀粉样蛋白斑块是AD的病理学标志。鉴于beclin 1在神经元中表达，beclin 1在哺乳动物中的这种新功能可能会影响对神经元健康重要的受体的再循环。 神经元的存活依赖于营养因子信号传导，并且在AD中已经观察到神经营养信号传导途径的改变，包括神经保护性TGF-β途径。我在这里提供的数据表明，在体外敲低beclin 1而不是自噬相关蛋白Atg 7，会降低TGF-β信号传导。已知Beclin 1通过与两种不同的III型磷脂酰肌醇-3-激酶（PI3K）复合物相互作用来介导其在酵母中的自噬和蛋白分选中的作用。PI3K的催化亚基的敲低导致TGF-β信号传导减少。此外，UVRAG（一种对酵母中的蛋白质分选特异性的PI3K复合物组分）的敲低也降低了TGF-β信号传导，而对参与自噬的复合物特异性的Atg14的敲低则没有影响。这表明beclin 1作为含有UVRAG的PI3K复合物的一部分在调节TGF-β信号传导中具有特定作用。 这项提议的目的是检验这样一个假设，即减少beclin 1有助于神经退行性变，部分是通过损害TGF-β受体再循环和剥夺神经元通过这一途径的保护性信号传导。在目的1中，我确定beclin 1和PI3K复合物之间的功能和物理相互作用，该复合物使用一系列突变体构建体介导TGF-β信号传导。我还通过活细胞成像表征了beclin 1、TGF-β受体和PI3-磷酸（PI3K复合物的产物）之间的关系。在目标2中，我使用流式细胞术、显微镜和免疫沉淀的组合来确定beclin 1在TGF-β信号通路中的功能，以直接测试beclin 1 KD对TGF-β受体内吞和再循环以及下游信号分子的募集的影响。在目标3中，我测试beclin 1 KD是否在体内损害TGF-β信号传导并阻止TGF-β介导的神经保护作用对抗兴奋性毒性。这些实验使用生物发光成像和免疫组织化学的组合来确定beclin 1 KD对TGF-β信号传导和神经变性的影响。 目前正在研究针对AD中TGF-β通路的治疗方法。考虑到beclin 1水平降低对TGF-β信号传导的影响，了解这一过程的机制至关重要，以便我们最大限度地提高成功开发这种毁灭性疾病疗法的机会。

DESCRIPTION (provided by applicant): Alzheimer Disease (AD) affects over 5 million people in the U.S. alone but our understanding of the mechanisms underlying pathogenesis remains incomplete and there is currently no cure. Recently, we reported that levels of the autophagy-related protein beclin 1 are decreased in AD brain extract. Heterozygous deficiency of beclin 1 in mice results in synaptic loss and neurodegeneration. Reduced beclin 1 levels in microglia impair recycling of the phagocytic receptor CD36, resulting in reduced clearance of amyloid plaques, a pathological hallmark of AD. Given that beclin 1 is expressed in neurons, this novel function of beclin 1 in mammals may affect recycling of receptors important for neuronal health. Neuronal survival depends on trophic factor signaling, and alterations in neurotrophic signaling pathways, including the neuroprotective TGF-¿ pathway, have been observed in AD. I present data here that shows knock-down of beclin 1, but not the autophagy-related protein Atg7, decreases TGF-¿ signaling in vitro. Beclin 1 is known to mediate its roles in autophagy and protein sorting in yeast through interaction with two distinct type III phosphatidylinositol-3-kinae (PI3K) complexes. Knock-down of the catalytic subunit of the PI3K results in decreased TGF-¿ signaling. Furthermore, knock-down of UVRAG, a PI3K complex component specific to protein sorting in yeast, also decreased TGF-¿ signaling, while knock-down of Atg14, specific to the complex involved in autophagy, has no effect. This suggests a specific role for beclin 1, as part of the PI3K complex containing UVRAG, in regulating TGF-¿ signaling. The purpose of this proposal is to test the hypothesis that reduced beclin 1 contributes to neurodegeneration, in part, by impairing TGF-¿ receptor recycling and depriving neurons of protective signaling through this pathway. In Aim 1, I determine the functional and physical interactions between beclin 1, and the PI3K complexes that mediate TGF-¿ signaling using a series of mutant constructs. I also characterize the relationship between beclin 1, the TGF-¿ receptors and PI3-phosphate, the product of the PI3K complex, by live-cell imaging. In Aim 2, I determine where in the TGF-¿ signaling pathway beclin 1 functions using a combination of flow cytometry, microscopy, and immunoprecipitation to directly test the effect of beclin 1 KD on TGF-¿ receptor endocytosis and recycling, and recruitment of downstream signaling molecules. In Aim 3, I test if beclin 1 KD impairs TGF-¿ signaling in vivo and prevents TGF-¿-mediated neuroprotection against excitotoxicity. These experiments use a combination of bioluminescence imaging and immunohistochemistry to determine the effect of beclin 1 KD on TGF-¿ signaling and neurodegeneration. Therapeutic approaches targeting the TGF-¿ pathway in AD are currently under study. Given the effect of reduced beclin 1 levels on TGF-¿ signaling, it is crucial to understand the mechanisms underlying this process so that we maximize our chance of success in developing therapies for this devastating disease.